Diagnostic and therapeutic methods based on the l1 adhesion molecule for ovarian and endometrial tumors

ABSTRACT

A method is described for the diagnosis of an ovarian or endometrial tumor or for a prognosis, characterized in that the L1 level is determined in a patient sample, preferably via an anti L1 antibody, the presence of L1 being an indication of the presence of an ovarian or endometrial tumor or a predisposition for such a tumor. Furthermore, methods for treating of ovarian or endometrial tumors are provided.

[0001] The present invention relates to a method for the diagnosis of anovarian or endometrial tumor or for a prognosis, characterized in thatthe L1 level is determined in a patient sample, the presence of L1 beingan indication of the presence of an ovarian or endometrial tumor or apossible predisposition for such a tumor. This detection is madepreferably via a monoclonal anti L1 antibody or a fragment thereof. Theinvention also relates to pharmaceutical preparations for use in thetherapy of an ovarian or endometrial tumor. Furthermore, the presentinvention concerns a method of treating ovarian or endometrial tumors.

[0002] The diagnosis of specific tumors is based inter alia on thedetection of specific tumor antigens (TAG) which may originate from thecytoplasm, the cell surface and the nucleus. In this connection, adistinction is made between tissue, individual and species-specific TAGswhich may also be present physiologically as differentiation antigens oncells and between TAGs which are referred to as tumor-specificneoantigens and TAGs as intermediate products of the oncogenesis of acell type which disappear again with further differentiation. They aredetectable as tumor markers by immunochemical methods, above allintracellular antigens (AG) or antigens forming (increasingly) assurface antigens on tumor cells being of interest. Of diagnosticsignificance are e.g. the oncofetal AG (“OFA”), e.g. thecarcinoembryonal AG (in the case of colon carcinoma), the SCC AG(“squamous cell carcinoma antigen”), _(α1) fetoprotein (in the case ofprimary liver cell carcinoma), isoferritin and fetal sulfoglycoprotein(in the case of gastric cancer and colon carcinoma), _(α2)-Hferroprotein (in the case of a malignant tumor occurring in earlychildhood), _(γ)-fetoprotein (in the case of sarcomas, leukemias, breastcancers), furthermore “Tennessee AG” (tennagen), “tissue polypeptide AG”(TPA), oncofetal membrane AG (OFMA), tumor-specific transplantation AG(TSTA), membrane-associated tumor AG (MATA) as well as minor antigenssuch as “A-like” AG, “Forssman” AG, WGL, etc. Another tumor marker useddiagnostically so far is CA125 (highly glycosylated cellular mucin whichoccurs in tumor cells to a greater degree and is released) which markerhas been used so far for the diagnosis of human ovarian cancer orendometrial carcinoma, e.g. via the determination of the serum levelsor—after operations—via the immunohistological staining of tissuesamples with anti CA125 antibodies. However, the diagnosis based on thedetection of CA125 has a number of serious drawbacks. For example,increased serum levels of CA125 can also occur in a number of benignantdiseases, e.g. in the case of an endometrial inflammation, pelvicinflammation, cirrhosis or also during the menstruation or a pregnancy.Increased CA125 levels are also found in the case of non-gynecologicmalignant tumors such as breast cancer, colorectal cancer, pancreaticcarcinoma or lung cancer. Thus, this can lead to false-positivediagnoses. CA125 is also found in the case of immunostaining ofcolon-adenocarcinomas, gastric cancer, lung cancer and atypicalhyperplasias of the endometrium. For the early detection of ovarian orendometrial carcinoma the measurement of CA125—in combination withultrasound—does not yield any satisfactory reliable results either.Thus, with respect to both susceptibility and specificity CA125 is notsuited for an early detection of ovarian or endometrial cancer but—if atall—rather for subsequent stages or relapses, however, the abovedescribed problems regarding the specificity existing, i.e. thediagnostically hardly acceptable occurrence of false-positive findings.

[0003] Therefore, the invention is based on the technical problem ofproviding markers which permit an improved and more specific diagnosisof ovarian and endometrial cancers, including the possible distinctionbetween various tumor types and the possibility of detecting metastasesand thus improved therapeutic, e.g. surgical, measures. A furthertechnical problem is to provide a therapeutic approach by using L1 astherapeutic target. The solution to this technical problem is achievedby providing the embodiments characterized in the claims.

[0004] It has surprisingly been found that the L1 adhesion moleculerepresents a highly specific marker in body fluids (e.g. in the bloodserum, ascites and fluid from Douglas coule de sac) and tissue fromovarian and endometrial tumors, above all for highly aggressive forms ofthese tumors. L1 is a neuronal adhesion molecule of 200-230 kDa, whichas regards its structure belongs to the Ig superfamily. L1 is a proteinassociated with cell migration, which in the brain is involved in theneuronal migration in the cerebellum and in the fasciculation ofneurites. L1 has several binding partners among them L1 itself, theproteoglycan neurocan and various integrins (Kadmon et al.,Differentiation 61: 143-150, 1997). Soluble L1 can stimulate themigration of tumor cells and hence promote metastasis (Mechtersheimer etal., JBC 2001, in press). In the screening experiments resulting in thepresent invention it turned out that L1 is not expressed in normaltissues with the exception of peripheral nerves and ganglions. Inbenignant ovarian and endometrial tumors no L1 expression could bedetected, and the screening of other malignant tumors (breast, prostate,cervix) was also negative. It showed that L1 is expressed on highlyaggressive serous papilloma carcinomas of the ovary and the endometrium.Soluble L1 was detectable in the serum and ascitic or pleural fluids oftumor patients but not in normal sera and body fluids or in patientshaving benignant tumors. Preoperative samples which were obtained bygynecologic curettage were positive in serous papilloma carcinomas ofthe endometrium and with the involvement of the ovary. Thus, theexpression of L1 as cell surface molecule in the serum and in bodyfluids is highly significant as regards the early detection ofaggressive ovarian and endometrial tumors and also as regards theprognostication. Therefore, the L1 detection permits the early detectionand accurate diagnosis or prognosis of aggressive ovarian andendometrial tumors. Thus, the subject matter of the present inventionrelates to a method for the diagnosis of an ovarian or endometrial tumoror for a prognosis, characterized in that the L1 level is determined ina patient sample, the presence of L1 being an indication of the presenceof an ovarian or endometrial tumor or a predisposition for such a tumor.

[0005] Suitable methods of obtaining a patient sample are known to aperson skilled in the art. The L1 adhesion molecule can be detected viacommon methods, the known amino acid sequence of the L1 protein or thenucleic acid sequence of the corresponding gene being used as a basis(Reid, R. A. et al., J. Mol. Neurosci. 3: 127-135, 1992). In thisconnection, the detection may refer to the transcription (detection ofthe concentration of the mRNA via common methods) or the L1 protein perse, the latter being preferred. The expression “the presence of L1 [as]an indication of the presence of an ovarian or endometrial tumor or apredisposition for such a tumor” also comprises the situation in whichthe concentration of L1 is increased as compared to a control (e.g. bodyfluid or tissue of a healthy person).

[0006] In a first preferred embodiment of the method according to theinvention the L1 level is determined by determining L1 as cell surfacemolecule in tissue. Suitable tissues are removed tumor tissue and cellmaterial obtained by curettage, the tissue obtained from a gynecologiccurettage being preferred.

[0007] In a second preferred embodiment of the method according to theinvention the L1 level is determined by determining L1 in soluble formin a body fluid. Suitable body fluids are blood serum, ascitic orpleural fluid or fluid from Douglas coule de sac, serum being preferred.

[0008] A third preferred embodiment does not carry out the detection ofL1 but the detection of L1-specific antibodies by means of (recombinant)L1. For this purpose, purified (recombinant) L1 is immobilized on thesurface of microtitration plates and then incubated with patient serum.Bound antibodies are then detected by means of an enzyme-conjugatedsecond antibody and a color reaction.

[0009] In a fourth preferred embodiment L1 mRNA is detected in a bodyfluid sample, preferably in blood serum.

[0010] Suitable detection methods of L1, L1 mRNA and L1 antibodies,respectively, and suitable specific probes are known to the personskilled in the art, the detection of L1 protein via specific antibodiesbeing preferred, the same or different antibodies being usable for boththe soluble form and for the form on the cell surface.

[0011] In the particularly preferred first and second embodiments of themethod according to the invention the L1 level is therefore determinedby contacting the patient sample with an anti-L1 antibody or a fragmentthereof and then determining whether the anti-L1 antibody or thefragment thereof has been bound to L1. In this regard reference is madeto FIG. 6A-C.

[0012] The antibodies suitable for this purpose may be monoclonal,polyclonal or synthetic antibodies or fragments thereof. In thisconnection, the expression “fragment” refers to all parts of themonoclonal antibody (e.g. Fab-, Fv- or “single chain Fv” fragments)which have the same epitope specificity as the complete antibody. Theproduction of such fragments is known to the person skilled in the art.The antibodies according to the invention are preferably monoclonalantibodies. The antibodies according to the invention can be preparedaccording to standard methods, L1 or a synthetic fragment thereof, whichis present in both L1 forms (soluble/anchored on the cell surface)preferably serving as an immunogen. This polypeptide or peptide and thefragment thereof, respectively, can be produced e.g. by obtaining thecorresponding gene, cloning and recombinant expression. Methods ofobtaining monoclonal antibodies are known to the person skilled in theart. Anti-L1 antibodies suitable for the diagnostic method according tothe invention are commercially available. These are e.g. the primaryhuman monoclonal anti L1 antibodies 15551A (Company of Pharmingen, SanDiego, USA) or MCA1753 (BIOZOL Diagnostica Vertrieb GmbH, Eching,Germany).

[0013] In an even more preferred embodiment of the method according tothe invention the L1 level is determined via a primary monoclonalanti-L1 antibody and a commercially available secondary antibody. Thesecondary antibody may carry any label which may be detected. It is,however, preferred that this secondary antibody may be biotin-conjugatedand may be detected by using streptavidin peroxidase. The person skilledin the art is perfectly familiar with methods of carrying out thisembodiment. The detection of L1 (e.g. via ELISA) can also achieved withonly one (monoclonal) antibody since L1 in serum is multimeric (c.f.Example 3, FIG. 5B).

[0014] In a preferred embodiment the patient sample is immobilized, e.g.as paraffin section. The patient sample can also be absorbed to the wallof a plastic dish by common methods such that L1 does not loose itsbonding specificity e.g. for a specific antibody added thereto.

[0015] In a preferred alternative embodiment of the method according tothe invention the anti L1 antibody or the fragment thereof isimmobilized, i.e. it is absorbed e.g. to the wall of a plastic dishwithout loosing its bonding specificity for L1. Then a patient sample isadded.

[0016] The bonding of the antibody can be detected by common methods,e.g. Western blot, ELISA, radioimmunoassay (RIA), etc., RIA and ELISAbeing preferred.

[0017] The above mentioned preferred fourth embodiment of the method ofthe present invention is based on the detection of L1 mRNA by RT-PCR. Ithas shown before that different isoforms of human L1 (i.e.hemoatopoietic L1 vs. neural L1) can be distinguished on the basis of L1exon 27 usage. Hematopoietic cells do not express exon 27. (Ebeling etal, Eur J Immunol. 26:2508-2516,1996). This exon encodes 4 aminoacids inthe cytoplasmic tail of L1. By RT-PCR using specific primer for thisregion of L1 it has now found out that ovarian tumors (established celllines and tumor speciments) express the neural form of L1 which ischaracterized by an insertion of 12 nucleotides (FIG. 4). Using the sameprimers it has been observed that L1 specific mRNA could be detected intumor patient sera. RT-PCR detection of tumor derived mRNA (possiblyderived from tumor cells in the blood) in addition to protein detectionby ELISA is a sensitive technique for diagnosis.

[0018] Based on the present invention a kit was also developed which isof use for the diagnostic method according to the invention andpreferably contains an anti L1 antibody or a fragment thereof and inaddition L1 or a bond-active part thereof for the purpose of control.The expression “bond-active part” refers to a fragment which reacts withthe antibody with which the whole molecule reacts as well. Depending onthe development of the kit, the antibody can be conjugated to anotherunit, e.g. a marker, and/or it can be immobilized on a solid carrier(substrate). The kit can also contain a second antibody for thedetection of L1/antibody complexes. The antibody or the fragment thereofmay be present in free form or be immobilized on a solid carrier, e.g. aplastic dish, a test tube, a microtitration plate, a test rod, etc. Thekit may also contain instructions which describe the use of the antibodyor a fragment thereof in an assay for the detection of a predispositionor the presence of a tumor. The kit can also contain suitable reagentsfor the detection of labelings or for the labeling of positive andnegative controls, wash solutions, dilution buffers, etc.

[0019] In the case of a positive diagnostic detection of an ovarian orendometrial tumor by means of the above described anti-L1 antibodies orfragments thereof, immunotherapy via the sensibilization oftumor-specific T lymphocytes can also be carried out according to knownmethods with the above mentioned antibodies or fragments thereof or withpeptides which are derived from L1 (on the basis of the known amino acidsequence) and have preferably a length of at least 9-12 amino acids. Inaddition, antibodies against L1 can also be used with the intention touse L1 as a tumor-specific target antigen.

[0020] Thus, the present invention also relates to a pharmaceuticalpreparation which contains an anti L1 antibody or a fragment thereof, ora peptide derived from L1, or a L1 antisense oligonucleotide.

[0021] The antibodies are preferably monoclonal antibodies. In aparticularly preferred embodiment said monoclonal antibody is anantibody originating from an animal (e.g. mouse), a humanized antibodyor a chimeric antibody or a fragment thereof. Chimeric antibodiesresembling human antibodies or humanized antibodies have a reducedpotential antigenicity, however, their affinity for the target is notlowered. The production of chimeric and humanized antibodies and ofantibodies resembling human antibodies, respectively, was described indetail (see e.g. Queen et al., Proc. Natl. Acad. Sci. USA 86 (1989),10029, and Verhoeyan et al., Science 239 (1988), 1534). Humanizedimmunoglobulins have variable framework regions which originatesubstantially from human immunoglobulin (with the designation ofacceptor immunoglobulin) and the complementarity of the determiningregions which originate substantially from a non-human immunoglobulin(e.g. of a mouse) (with the designation of donor immunoglobulin). Theconstant region(s) originate(s), if present, also substantially from ahuman immunoglobulin. When administered to human patients, humanized(and the human) antibodies offer a number of advantages over antibodiesof mice or other species: (a) the human immune system should not regardthe framework or the constant region of the humanized antibody asforeign and therefore the antibody response against such an injectedantibody should be less than against a completely foreign mouse antibodyor a partially foreign chimeric antibody; (b) since the effector regionof the humanized antibody is human, it interacts better with other partsof the human immune system, and (c) injected humanized antibodies have ahalf life which is substantially equivalent to that of naturallyoccurring human antibodies, which permits to administer smaller and lessfrequent doses as compared to antibodies of other species.

[0022] In a preferred embodiment thereof, L1 mAb is labeled with (123)Ifor imaging and injected i.p. or i.v. as described(vanZanten-Przybysz,I. Int.J.Cancer 92:106-114, 2001). MAb to L1 isinjected after stable conjugation with cytotoxic drugs in order to killtumor cells. Examples of such cytotoxic compounds are radionuclides,toxic proteins (e.g. saporin), chemotherapeutic agents or furtherantibodies with other specificity (e.g. against T cells, e.g. CD3), sothat bifunctional antibodies form which effect the killing of tumorcells by means of cytotoxic T cells.

[0023] In another preferred embodiment, overexpression of L1 in ovarianand endometrium tumors can be used to prime cytotoxic T lymphocytes inorder to kill tumor cells in situ. A) Peptides derived from the sequenceL1 may be used to vaccinate tumor patients. Preferably, a mixture ofL1-peptides restricted by HLA-A1 (ATEGWFIGF and GSDDSLADY), HLA-A2(LLANAYIYV and WLDEDGTTV) and HLA-A3 (VLTGYVLSY) in an emulsion withGM-CSF and Montanide ISA-51 adjuvant ((Seppic, Fairfield, N.J., USA) areused. Patients receive a vaccine comprising the 5 L1 peptides (50-200μg, preferably 100 μg each) and 150-200 μg (preferably 190 μg) of theHLA-DR-restricted tetanus helper peptide AQYIKANSKFIGITEL. This peptiderepresents peptide p2 of tetanus toxoid (residues 830-844) plus anamino-terminal alanine residue to prevent formation of pyroglutamatefrom the N-terminal glutamine residue. Tetanus peptide p2 is apromiscuous binder to HLA-DR molecules. Vaccines are administered with150-250 mg (preferably 225 μg) GM-CSF in Montanide ISA-51 adjuvant. Eachpatient is preferably immunized at days 0, 7, 14, 28, 35 and 42 for atotal of 6 immunizations as described by Yamshchikov et al Int.J.Cancer92:703 (2001). The individual time frame, doses and total number ofimmunizations can be determined by a medical doctor depending on thekind of tumor, tumor growth, presence/absence of metastasis andpatient's data (weight, age etc.). B) Dendritic cells derived from tumorpatients may be pulsed with the above mentioned L1 peptides as decribedby Lau et al J.Immunother 24:66-78 (2001). The dendritic cells arepreferably obtained by incubation of plastic-adherent peripheral bloodmononuclear cells from tumor patients with IL-4 and GM-CSF for 8 days inserumless medium. The dendritic cells are then pulsed with L1 peptides(each at approximately 50 μg/ml) for overnight and then used for(intravenous) infusions.

[0024] In a further preferred embodiment, the overexpression of L1 inovarial or endometrial tumors is treated with L1 antisenseoligonucleotides. Since cell migration is a prerequisite of tumor celldissemination it is adviseable to eliminate L1 from the tumor cells.This can be achieved by established antisense technology. An L1antisense oligonucleotide is used to downregulate L1 expression in tumorcells. It corresponds preferably to the sequence AGGCTGTCGTCACTGCCCA andis the inverse complement of nucleotides +3593/3611 of the sequence ofhuman L1. The phosphorothioate oligo-nucleotide is preferably injectedinto the peritoneum of the tumor patient at doses between 1-12 mg/kg toachieve significant concentrations of oligonucleotide. The individualtime frame, doses and total number of administrations can be determinedby a medical doctor depending on the kind of tumor, tumor growth,presence/absence of metastasis and patient's data (weight, age etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1A: Endometroid ovarian adenocarcinoma composed of tubularglands and lined by non-mucin secreting stratified epithelium.Immunohistochemical staining with anti L1 antibody shows strongheterogenous membral staining.

[0026] B: Serous carcinoma of the ovary showing papillary and slit likeconfigurations with solid tumor masses in the stroma. Strong positiveimmunohistochemical staining with anti L1.

[0027] C: Omentum with disseminated tumoral glands and desmoplasticreaction. Strong homogeneous immunostaining of tumoral cells by anti L1antibody.

[0028] D: Appendical wall infiltrated by ovarian carcinoma cells. Stronghomogeneous staining of tumoral cells by anti L1 antibody. No stainingof normal mucosal glands and lymphatic tissue. Small peripheral nervebundles show strong positive staining.

[0029] E: Vaginal wall showing small tumoral mass in the lamina propriaand small tumor emboli in the dilated lmyphatics. Immunostaining withanti L1 shows strong homogeneous staining.

[0030] F: Ovarian carcinoma embolus in the lymphatics of the myometriumshowing hetergeneous membranous staining of tumoral cell by anti L1.

[0031]FIG. 2: Detection of L1 from curettage material

[0032] A: Tissue obtained from curettage

[0033] B: Control sections from ovarian adenocarcinoma

[0034]FIG. 3: Detection of soluble L1 in body fluids (1F-14F) and sera(1S-32S) by means of ELISA. The samples 4F, 5F, 8F, 12F and 23Foriginate from female ovarian cancer patients.

[0035]FIG. 4: 1%-Agarose gel after PCR analysis of exon 27 usage mRNAsfrom ovarian tumor speciments or known tumor cell lines (AR, OAW, Mel63)were isolated and transcribed in cDNA. These cDNAs were used as templatefor PCR analysis of exon 27 usage, Plasmids encoding both forms wereused as control (−RSLE/+RLSE).

[0036]FIG. 5: Comparison of two different ELISA formats

[0037] A.: Microtiter plates were coated with L1 mAB 1 followed byblocking with 3% BSA/TBS and incubation with patient sera as describedin Example 2. Bound soluble L1 was detected using biotinylated L1 mABfollowed by Streptavidin-Peroxidase. This ELISA format can detectdimeric (multimeric) and monomeric L1.

[0038] B. The ELISA was carried out as described in A but bound solubleL1 was detected using L1 mAB 1 followed by Streptavidin-Peroxidase. ThisELISA format can detect only dimeric (multimeric) L1.

[0039]FIG. 6: A: Ovarian tumor specimens were lysed and analysed bySDS-PAGE and Western blotting using an antibody to the cytoplasmicportion of L1

[0040] B: Sera from tumor patients were immunoprecipitated with L1antibody coupled to sepharose, separated by SDS-PAGE and analysed byWestern blotting using the same anti L1 antibody

[0041] C: Sera of tumor patients and normal individuals were analysed byELISA for the presence of soluble L1. The number of patients from eachgroups is indicated in brackets.

[0042] The following examples explain the invention.

EXAMPLE 1

[0043] The Examination of Curettage Tissue Shows a High Correlationbetween the Expression of L1 and the Clinical Stage and the PathologicalDegree of the Tumors, Respectively

[0044] Tumor tissue is embedded in paraffin according to standardmethods and serial sections are made. Following the treatment of thesections in the microwave oven (10 min., 92° C.) in the presence of 1mmol EDTA (pH 8.0) immunostaining of the tissue is carried out by meansof the MCA1753 antibody (Biozol Diagnostica Vertiebs GmbH). The boundfirst antibody is detected by means of an enzyme-coupled second antibody(Vector ABC Kit; www.vectorlabs.com). Some exemplary stainings are shownin FIG. 1. The clinical data are listed in Tables 1 and 2.

[0045]FIG. 2 shows that L1 can also be detected by means of curettagetissue. Using the above described procedure, a similar staining patternas on the tumor sections results. The determination of L1 by means ofcurettage material permits an early preoperative classification of thetumor within the meaning of Tables 1 and 2.

EXAMPLE 2

[0046] ELISA Shows the Presence of Soluble L1 in sera and Ascitic Fluidof Tumor Patients, a Correlation between the Presence of L1 and theTumor Type (Ovarian and Endometrial Tumor) being Observed.

[0047] Samples of body fluids (ascites, serum) were tested for thepresence of soluble L1 using a “capture” ELISA. For this purpose,microtitration plates were coated with the human anti L1 antibody(concentration: 1 μg/ml) described in Example 1 and then a blocking stepwas carried out with 3% BSA in PBS (45 min., room temperature) toeliminate the non-specific bonding to the plate. The body fluid wasadded at differing concentrations (1:2 and 1:10 of the fluid in 3% BSAin PBS), and incubation was carried out at room temperature for 1 hour.Therefore, four wash steps followed in Tris-buffered common saltsolution (TBS, pH 8.0, in the presence of 0.02% Tween-20). Bound solubleL1 was determined by the addition of human biotin-conjugated anti L1antibody. For this purpose, the biotinylated antibody MCA1753 is addedto the microtitration plates for 1 hour. This was followed by 4 washsteps as described above. Another incubation at room temperature for 1hour with peroxidase-conjugated streptavidin (Dianova Hamburg, Germany)follows. Finally, another 4 wash steps are carried out. Hereinafter thesubstrate solution is added to the microtitration plates and uponappearance of the color reaction the result is evaluated on the ELISAreader. The results are shown in FIG. 3.

EXAMPLE 3

[0048] A New ELISA Format for the Detection of Soluble L1

[0049] The ELISA described above in Example 2 used the coating of themicrotiter plate with L1 mAb 1 (capturing monoclonal antibody) and thedetection of soluble L1 with a biotinylated (or otherwise labelled) L1mAb 2 (detecting monoclonal antibody). This type of ELISA is termed G/Kformat. We have now developed another format in which we use both forcapturing and detection the same mAb to L1 (K/K format). In FIG. 5A andB a comparison of both types of ELISA is shown using a positive serum(CA526, ovarian tumor patient) and seval control sera from unrelatedtumors. It is obvious that the new ELISA format in FIG. 5B gives abetter signal to noise ratio (appr. 5-8 fold) than the previous formatshown in FIG. 5A. As the K/K format can only work when the antibodyepitope for detection is not blocked by the antibdy used for capturing,these results implicate that in the serum sample the soluble L1 isdimeric or multimeric. The control data performed in FIG. 5 with bothtypes of ELISA using monomeric and dimeric L1 (the L1-Fc fusion proteinis dimeric) support this notion.

EXAMPLE 4

[0050] PCR Analysis for Detecting L1 mRNA in Tumor Samples

[0051] Primer sequences for PCR analysis are deduced from the human L1cDNA sequence (EMBL accession number M 74387). For the detection ofhuman mRNA encoding L1 in tumor tissue or patient serum samples mRNA isisolated using commercial kits (Roche Molecular Biochemicals) andtranscribed into cDNA. A nested PCR approach is used with the followingprimer combinations: First amplification primer 1: ACTGAGGGCTGGTTCATC(sense), primer 2: CTTGCACTGTACTGGCCA (antisense) (one cycle 45 sec, 94°C., 30 cycles of 1 min at 94° C., 1 min at 56° C., 1 min at 72° C.). Forthe second PCR 1 μl of the first PCR reaction is used with primer 1:ACTCAGTGAAGGATAAGGAG (sense), primer 2: TTGAGCGATGGCTGCTGCT. In analternative protocol the following primers are used: primer 1:AGGTCCCTGGAGAGTG (sense); primer 2: TTGAGCGATGGCTGCTGCT (antisense). Thetemperature profile for the PCR reaction are as described above. PCRproducts are preferably separated on a 2% agarose gel containing 0.5μg/ml ethidium bromide. As an alternative, the gel is blotted andhybridized with the labeled L1 oligonucleotide probe(TCTGAGGCCCGACCGATGAAAGATGAGACCTTC) by southern blotting to increase thesensitivity. TABLE 1 Ovarian carcinomas Seeding to Seeding DistantFollow Case Histological Histological serosal to Metastase Clinical upL1 No type grade surface omentum S stage (month) Reccurance Outcomestaining 1 Serous moderate-poorly + + − IIIc 9 AWD + 2 poorly + + LungIIIc/IV 1 DOD + Breast 3 ″ moderate-poorly + + Brain IV 24 + DOD + 4 ″moderate-poorly − + − IIIc 36 + DOD + 5 ″ poorly + + − IIIc 26 + DOD + 6″ moderate-poorly − + − IIIc/IV 35 + DOD + 7 ″ moderate-poorly + + −IIIc 22 + DOD + 8 ″ well + − − Ic 22 − NED − 10 ″ poorly − + − IIIc 62 +DOD + 11 ″ poorly + + Lung Liver IIIc/IV 18 + DOD + 13 ″ poorly − − − Ic77 − NED − 14 ″ poorly + − Liver IIIc 5 − AWD + 15 ″ poorly + − − IIIc11 − AWD + 16 ″ moderate + − III No information + 24 ″ moderate + − LungIV 21 + AWD + 17 Endometroid moderate − − − Ic 20 − NED − 18 ″ poorly −− − Ic 54 − NED − 19 ″ poorly − − − Ic 55 − NED − 20 ″ moderate − − − Ic26 − NED − 21 ″ moderate − − − Ia 45 − NED − 22 ″ poorly − − − Ib 38 −NED − 23 ″ moderate − + − IIIc 6 − AWD + 25 ″ moderate + + − IIIc Noinformation + 26 ″ poorly + − Liver IIIc 35 − DOD + 27 ″ moderate − −Vagina IIIc 12 + DOD + 28 ″ poorly + + Lung IV 20 − AWD + 29 ″poorly + + − IIIc 17 + DOD + 30 ″ poorly + + Lung IV 6 + AWD + 31 ″moderate-poorly + + − IIIc No information + 32 ″ moderate + + Ic 35 −NED +

[0052] TABLE 2 Uterine carcinomas Seeding to serosal surface Follow CaseHistological Histological and Adnexal Clinical up L1 staining No typegrade omentum involvement stage (month) Recurrance Outcome curretageendometrium ovary *1 Endometroid poorly + + IIc ? + DOD ND + + 2 ″moderate + + IV 21 + DOD ND + + 3 Serous poorly + + IIIc Noinformation + + + 4 ″ poorly + + III ″ + + + 5 ″ moderate + + * ″ ND + +6 ″ poorly + + * ″ + + + 7 ″ poorly + + IV 10 + DOD ND + + 8 ″poorly + + IV  5 + DOD + + + 9 ″ poorly + + IIIc 26 + DOD ND + + 10 ″moderate-poorly + + IIIc 22 + DOD ND + + 11 ″ poorly − − Ib  6 − NED − −− 12 ″ moderate poorly − − Ic 28 − NED − − −

[0053]

1 13 1 9 PRT Artificial Sequence Synthetic Construct 1 Ala Thr Glu GlyTrp Phe Ile Gly Phe 1 5 2 9 PRT Artificial Sequence Synthetic Construct2 Gly Ser Asp Asp Ser Leu Ala Asp Tyr 1 5 3 9 PRT Artificial SequenceSynthetic Construct 3 Leu Leu Ala Asn Ala Tyr Ile Tyr Val 1 5 4 9 PRTArtificial Sequence Synthetic Construct 4 Trp Leu Asp Glu Asp Gly ThrThr Val 1 5 5 9 PRT Artificial Sequence Synthetic Construct 5 Val LeuThr Gly Tyr Val Leu Ser Tyr 1 5 6 16 PRT Artificial Sequence SyntheticConstruct 6 Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr GluLeu 1 5 10 15 7 19 DNA Artificial Sequence Synthetic Construct 7aggctgtcgt cactgccca 19 8 18 DNA Artificial Sequence Synthetic Construct8 actgagggct ggttcatc 18 9 18 DNA Artificial Sequence SyntheticConstruct 9 cttgcactgt actggcca 18 10 20 DNA Artificial SequenceSynthetic Construct 10 actcagtgaa ggataaggag 20 11 19 DNA ArtificialSequence Synthetic Construct 11 ttgagcgatg gctgctgct 19 12 16 DNAArtificial Sequence Synthetic Construct 12 aggtccctgg agagtg 16 13 33DNA Artificial Sequence Synthetic Construct 13 tctgaggccc gaccgatgaaagatgagacc ttc 33

1. A method for the diagnosis of an ovarian or endometrial tumor or fora prognosis, characterized in that the L1 level is determined in apatient sample, the presence of L1 being an indication of the presenceof an ovarian or endometrial tumor or a predisposition for such a tumor.2. The method according to claim 1, wherein the L1 level is determinedby determining L1 as a cell surface molecule in tissue.
 3. The methodaccording to claim 1, wherein the L1 level is determining by detectingL1 in soluble form in a body fluid.
 4. The method according to claim 3,wherein the body fluid comprises serum.
 5. The method according to claim1, wherein the L1 level is determined by contacting the patient samplewith an anti L1 antibody or a fragment thereof and then determiningwhether the anti L1 antibody or the fragment thereof has been bound toL1.
 6. The method according to claim 5, which is carried out as aradioimmunoassay or ELISA.
 7. The method according to claim 1, whereinthe L1 level is determined by determining the presence of anti L1antibodies or a fragment thereof in the patient sample by means ofbonding to (recombinant) L1.
 8. The method according to claim 1, whereinthe L1 level is determined by determining the presence of L1 mRNA in abody fluid.
 9. The method of claims 8, wherein said determining stepcomprises RT-PCR.
 10. A pharmaceutical preparation, containing an antiL1 antibody or a fragment thereof.
 11. A pharmaceutical preparation,containing a peptide derived from L1 or a fragment thereof.
 12. Apharmaceutical preparation, containing a L1 antisense oligonucleotide.13. Use of an anti L1 antibody or a fragment thereof for treatingovarian or endometrial tumors.
 14. Use of a peptide derived from L1 or afragment thereof for treating ovarian or endometrial tumors.
 15. Use ofa L1 antisense oligonucleotide for treating ovarian or endometrialtumors.
 16. A method of treating ovarian or endometrial tumors in apatient in need of such treatment, said method comprising the step ofadministering to the patient a sufficient amount of a L1 antibody orfragment thereof conjugated to a cytotoxic drug.
 17. A method oftreating ovarian or endometrial tumors in a patient in need of suchtreatment, said method comprising the step of administering to thepatent a sufficient amount of a protein derived from L1 or a fragmentthereof in a vaccination preparation.
 18. A method of treating ovarianor endometrial tumors in a patient in need of such treatment, saidmethod comprising the step of administering to the patient a sufficientamount of a L1 antisense oligonucleotide.
 19. The method according toclaim 3, wherein the body fluid comprises a fluid selected from thegroup consisting of blood serum, ascites, pleural fluid, and Douglascoule de sac fluid.
 20. A method of screening a patient for ovarianand/or endometrial carcinoma, comprising determining whether L1 ispresent in the patient that is non-attributable to expression of L1 inperipheral nerves and ganglions of the patient.
 21. The method accordingto claim 20, wherein said carcinoma comprises a serous papillomacarcinoma, and said determining step comprises analyzing a fluid fromthe patient, wherein said fluid is selected from the group consisting ofserum, Douglas coule de sac, ascitic and pleural fluids.
 22. The methodaccording to claim 20, wherein said determining step comprises analyzingan ovarian tissue sample of the patient.
 23. A kit for screening apatient for ovarian and/or endometrial tumor comprising: (a) an anti L1antibody or fragment thereof; (b) L1 or a bond active part thereof, as acontrol; and (c) instructions describing use of (a) in an assay fordetermining of presence of or predisposition to said tumor.
 24. Thepharmaceutical preparation of claim 10, wherein said L1 antibody orfragment thereof is conjugated to a cytotoxic agent.